Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)

Place the vial in the hood and clean it with 70% ethanol

Immediately remove the contents of the vial and place into the cold media

Rinse the tube down with some of the cold media from the second vial

Spin down cells at 2000 rpm (80-100 x g) for 5 min

Aspirate off the cold media and resuspend the cells in the warm media

Transfer the cells and the media into a petri dish

Count cells

Place in incubator

Change the media once the cells have attached to the plate

Removes remaining DMSO

Allow cells to grow to 80-90% confluency

Split cells (1:3) into petri dishes

Continue to split cells and freeze down as needed

Notes

List troubleshooting tips here.

You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.

Anecdotal observations that might be of use to others can also be posted here.

Alternative Thawing

From step 5 above, using a 1 ml pipet add 1 ml of the cold media from the 15 ml tube drop by drop to the vial (about 1 drop every 10 seconds).

Plate immediately with the cold media and change the media once cells have attached (12 - 24 hours later) to remove DMSO that was in the freezing media. Usually a 1:3 ratio is good for MC3T3s and a 1:4 ratio for MLOY4s.

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